HCV NS3 recombinant antigens and mutants thereof for improved antibody detection

Abstract

The present disclosure relates to polypeptides, including fusions thereof, nucleic acids, vectors, host cells, immunodiagnostic reagents, kits, and immunoassays for use detecting the presence of HCV antibodies. More specifically, the present invention describes specific NS3 antigens that can be used for the detection of anti-HCV antibodies.

The invention claimed is: 
     
       1. A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I, II, and III of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from test sample as compared to C33 antigen, wherein said recombinant HCV NS3 antigen comprises one or more of the characteristics selected from the group consisting of:
 diminished ATP-binding activity as compared to the ATP-binding activity of wild-type NS3 helicase; 
 diminished ATPase activity as compared to wild-type NS3 helicase; and 
 increased redox stability as compared to the redox stability of wild-type NS3 helicase, 
 wherein said wild-type HCV NS3 helicase comprises the amino acid sequence of SEQ ID NO: 87, wherein the C33 antigen corresponds to positions 1192-1457 of SEQ ID NO: 88, and 
 wherein said antigen comprises the amino acid sequence of SEQ ID NO:87 comprising at least one of the following mutations: 
 (a) a mutation of one or more cysteine residues to a corresponding serine residue, the one or more cysteine residues selected from the group consisting of C368, C374, C499, and C525 of wild-type HCV NS3 protein which correspond to C203, C209, C334, and C360 of SEQ ID NO: 87, wherein the wild-type HCV NS3 protein comprises the sequence of SEQ ID NO: 87; or 
 (b) a mutation that diminishes ATP binding or diminishes ATPase activity comprising a replacement of one or more of the amino acid residues with any other amino acid residue, the one or more of the amino acid residues selected from the group consisting of S211, T212, Y241, and T419 of wild-type HCV NS3 protein which correspond to S46, T47, Y76, and T254 of SEQ ID NO: 87. 
 
     
     
       2. The recombinant HCV NS3 antigen of  claim 1 , wherein said antigen further comprises addition of at least one cysteine residue to the C-terminal end of said NS3 helicase. 
     
     
       3. The recombinant HCV NS3 antigen of  claim 1 , wherein said mutation comprises a replacement of one or more of the amino acid residues with any other amino acid residue, the one or more of the amino acid residues selected from the group consisting of S211, T212, Y241, and T419 of wild-type HCV NS3 protein which correspond to S46, T47, Y76, and T254 of SEQ ID NO: 87. 
     
     
       4. The recombinant HCV NS3 antigen of  claim 1 , wherein said mutation comprises a mutation of one or more of the cysteine residues selected from the group consisting of C368, C374, C499, and C525 of wild-type HCV NS3 protein which correspond to C203, C209, C334, and C360 of SEQ ID NO:87. 
     
     
       5. The recombinant HCV NS3 antigen of  claim 4 , wherein said antigen further comprises addition of at least one cysteine residue to the C-terminus end of said NS3 helicase. 
     
     
       6. The recombinant HCV NS3 antigen of  claim 3 , wherein said antigen further comprises addition of at least one cysteine residue to the C-terminus end of said NS3 helicase. 
     
     
       7. The recombinant HCV NS3 antigen of  claim 6 , wherein said addition of a cysteine residue to the C-terminal end of said NS3 helicase comprises addition of a sequence selected from the group consisting of GGCSGGA (SEQ ID NO: 82), DECHSTD (SEQ ID NO: 84), and SKKKCDE (SEQ ID NO: 86) to the C-terminal end of said NS3 helicase, optionally further comprising conjugation to a signal generating moiety. 
     
     
       8. A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I, II, and III of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from test sample as compared to C33 antigen, wherein said recombinant HCV NS3 antigen comprises one or more of the characteristics selected from the group consisting of:
 diminished ATP-binding activity as compared to the ATP-binding activity of wild-type NS3 helicase; 
 diminished ATPase activity as compared to wild-type NS3 helicase; and 
 increased redox stability as compared to the redox stability of wild-type NS3 helicase, 
 wherein said wild-type HCV NS3 helicase comprises the amino acid sequence of SEQ ID NO: 87, wherein the C33 antigen corresponds to positions 1192-1457 of SEQ ID NO: 88, and 
 wherein said antigen comprises the amino acid sequence of SEQ ID NO:87 comprising at least one mutation that diminishes ATP binding or diminishes ATPase activity and is a replacement of one or more of the amino acid residues with any other amino acid residue, the one or more of the amino acid residues selected from the group consisting of K210, S211, T212, Y241, D290, E291, H293, T419, Q460, R464, R467, and W501 of wild-type HCV NS3 protein which correspond to K45, S46, T47, Y76, D125, E126, H198, T254, Q295, R299, R302, and W366 of SEQ ID NO:87 wherein the wild-type HCV NS3 protein comprises the sequence of SEQ ID NO: 87, 
 wherein said antigen further comprises addition of at least one cysteine residue to the C-terminal end of said NS3 helicase, and 
 wherein said addition of at least one cysteine residue to the C-terminal end of said NS3 helicase comprises addition of a sequence selected from the group consisting of 
 GSGSGHHHHHHHHGGCSGGARSGC (SEQ ID NO: 89); 
 GSGSGHHHHHHHHDECHSTDRSGC (SEQ ID NO: 90); and 
 GSGCGHHHHHHHHGGCSGGA (SEQ ID NO: 91), optionally further comprising conjugation to a signal generating moiety. 
 
     
     
       9. The recombinant HCV NS3 antigen of  claim 6 , wherein said antigen further comprises a histidine tag. 
     
     
       10. The recombinant HCV NS3 antigen of  claim 9 , wherein said histidine tag is located between the C-terminus of SEQ ID NO:87 and the N-terminus of said added sequence. 
     
     
       11. The recombinant HCV NS3 antigen  claim 1 , wherein said antigen is biotinylated either at the N-terminus, the C-terminus or at a site specific biotinylation distal from the C or N terminus of said antigen. 
     
     
       12. An isolated nucleic acid encoding a recombinant HCV antigen of  claim 1 . 
     
     
       13. An expression vector comprising an isolated nucleic acid of  claim 12 . 
     
     
       14. A host cell transformed or transfected with an expression vector of  claim 13 . 
     
     
       15. An immunodiagnostic reagent comprising the recombinant HCV antigen of  claim 1 . 
     
     
       16. The immunodiagnostic reagent of  claim 15 , further comprising a solid support. 
     
     
       17. The immunodiagnostic reagent of  claim 15 , wherein said recombinant antigen is detectably labeled with a fluorescent label. 
     
     
       18. A kit comprising an immunodiagnostic reagent of  claim 15  and further comprising an additional isolated HCV antigen comprising an epitope that is immunoreactive with an anti-HCV antibody. 
     
     
       19. The kit of  claim 18 , wherein said recombinant HCV NS3 antigen and said additional HCV antigen are either co-coated on the same solid phase, or are each coated on a separate solid phase. 
     
     
       20. The kit of  claim 19 , further comprising antibodies for detection of human antibodies. 
     
     
       21. The kit of  claim 19 , further comprising anti-HCV antibodies, optionally comprising a detectable label. 
     
     
       22. An immunoassay method of determining the presence of anti-HCV antibodies in a test sample, comprising contacting said test sample with an immunodiagnostic agent of  claim 15  under conditions to allow a complex to from between said recombinant HCV NS3 antigen and said anti-HCV antibodies in said test sample, and detecting the presence of said complex, wherein presence of said complex is indicative of anti-HCV antibodies in said test sample. 
     
     
       23. The immunoassay method of  claim 22 , wherein said detection of said complex formation is detected by determining binding of labeled anti-human antibodies to said complex, wherein said anti-human antibodies are labeled with a fluorescent label. 
     
     
       24. The immunoassay method of  claim 22 , wherein the recombinant HCV NS3 antigen is coated on microparticles. 
     
     
       25. The immunoassay method of  claim 22 , wherein said method further comprises assaying said test sample to determine the presence of antibodies against HCV core antigen, optionally wherein said recombinant HCV NS3 antigen and said HCV core antigen are co-coated on the same microparticle or are coated on separate microparticles. 
     
     
       26. The immunoassay method of  claim 25 , wherein the test sample was obtained from a patient and the method further comprises diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of the patient, wherein, if the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment of the patient, the method optionally further comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve efficacy. 
     
     
       27. The immunoassay method of  claim 25 , wherein the method is adapted for use in an automated system or a semi-automated system. 
     
     
       28. The recombinant HCV NS3 antigen of  claim 1 , wherein said recombinant HCV NS3 antigen comprises increased redox stability as compared to the redox stability of wild-type NS3 helicase. 
     
     
       29. The recombinant HCV NS3 antigen of  claim 8 , wherein said antigen further comprises a mutation of one or more of the cysteine residues to any other amino acid, the one or more of the cysteine residues selected from the group consisting of C292, C368, C374 C499, and C525 of wild-type HCV NS3 protein which correspond to C127, C203, C209, C334, and C360 of SEQ ID NO: 87.